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Zn 2+ , Mg 2+ and Ca 2+ are essential divalent cations implicated in many metabolic processes and signalling pathways. An emerging new paradigm is that the organismal balance of these cations predominantly depends on a common gatekeeper, the channel-kinase TRPM7. Despite extensive electrophysiological studies and recent cryo-EM analysis, an open question is how the channel activity of TRPM7 is activated. Here, we performed site-directed mutagenesis of mouse TRPM7 in conjunction with patch-clamp assessment of whole-cell and single-channel activity and molecular dynamics (MD) simulations to show that the side chains of conserved N1097 form an inter-subunit Mg 2+ regulatory site located in the lower channel gate of TRPM7. Our results suggest that intracellular Mg 2+ binds to this site and stabilizes the TRPM7 channel in the closed state, whereas the removal of Mg 2+ favours the opening of TRPM7. Hence, our study identifies the structural underpinnings through which the TRPM7 channel is controlled by cytosolic Mg 2+ , representing a new structure-function relationship not yet explored among TRPM channels., (© 2022. The Author(s).)

Transient receptor potential A1 (TRPA1) channels were originally characterized in neuronal tissues but also identified in lung epithelium by staining with fluorescently coupled TRPA1 antibodies. Its exact function in non-neuronal tissues, however, is elusive. TRPA1 is activated in vitro by hypoxia and hyperoxia and is therefore a promising TRP candidate for sensing hyperoxia in pulmonary epithelial cells and for inducing alveolar epithelial hyperplasia. Here, we isolated tracheal, bronchial, and alveolar epithelial cells and show low but detectable TRPA1 mRNA levels in all these cells as well as TRPA1 protein by Western blotting in alveolar type II (AT II) cells. We quantified changes in intracellular Ca 2+ ([Ca 2+ ] i ) levels induced by application of hyperoxic solutions in primary tracheal epithelial, bronchial epithelial, and AT II cells isolated from wild-type (WT) and TRPA1-deficient (TRPA1-/-) mouse lungs. In all cell types, we detected hyperoxia-induced rises in [Ca 2+ ] i levels, which were not significantly different in TRPA1-deficient cells compared to WT cells. We also tested TRPA1 function in a mouse model for hyperoxia-induced alveolar epithelial hyperplasia. A characteristic significant increase in thickening of alveolar tissues was detected in mouse lungs after exposure to hyperoxia, but not in normoxic WT and TRPA1-/- controls. Quantification of changes in lung morphology in hyperoxic WT and TRPA1-/- mice, however, again revealed no significant changes. Therefore, TRPA1 expression does neither appear to be a key player for hyperoxia-induced changes in [Ca 2+ ] i levels in primary lung epithelial cells, nor being essential for the development of hyperoxia-induced alveolar epithelial hyperplasia.

Analysis of G-protein-coupled receptor (GPCR) signaling, in particular of the second messenger cAMP that is tightly controlled by G s - and G i/o -proteins, is a central issue in biomedical research. The classical biochemical method to monitor increases in intracellular cAMP concentrations consists of a radioactive multicellular assay, which is well established, highly sensitive, and reproducible, but precludes continuous spatial and temporal assessment of cAMP levels in single living cells. For this purpose, Förster resonance energy transfer (FRET)-based Epac cAMP sensors are well suitable. So far, the latter sensors have been employed to monitor G s -induced cAMP increases and it has remained elusive whether Epac sensors can reliably detect decreased intracellular cAMP levels as well. In this study, we systematically optimize experimental strategies employing FRET-based cAMP sensors to monitor G i/o -mediated cAMP reductions. FRET experiments with adrenergic α 2A or μ opioid receptors and a set of different Epac sensors allowed for time-resolved, valid, and reliable detection of cAMP level decreases upon G i/o -coupled receptor activation in single living cells, and this effect can be reversed by selective receptor antagonists. Moreover, pre-treatment with forskolin or 3-isobutyl-1-methylxanthine (IBMX) to artificially increase basal cAMP levels was not required to monitor G i/o -coupled receptor activation. Thus, using FRET-based cAMP sensors is of major advantage when compared to classical biochemical and multi-cellular assays.

Transient receptor potential cation channel, subfamily M, member 7 (TRPM7) is a bi-functional protein comprising an ion channel moiety covalently linked to a protein kinase domain. Currently, the prevailing view is that a decrease in the cytosolic Mg(2+) concentration leads to activation of divalent cation-selective TRPM7 currents. TRPM7 plays a role in immune responses, hypotension, tissue fibrosis, and tumor progression and, therefore, represents a new promising therapeutic target. Because of the dearth of pharmacological tools, our mechanistic understanding of the role of TRPM7 in physiology and pathophysiology still lags behind. Therefore, we have recently carried out a high throughput screen for small-molecule activators of TRPM7. We have characterized the phenanthrene naltriben as a first stimulatory agonist of the TRPM7 channel. Surprisingly, the effect of naltriben on TRPM7 was found to be unaffected by the physiological levels of cytosolic Mg(2+). Here, we demonstrate that mibefradil and NNC 50-0396, two benzimidazole relatives of the TRPM7 inhibitor NS8593, are positive modulators of TRPM7. Using Ca(2+) imaging and the patch-clamp technique, we show that mibefradil activates TRPM7-mediated Ca(2+) entry and whole-cell currents. The response to mibefradil was fast, reversible, and reproducible. In contrast to naltriben, mibefradil efficiently activates TRPM7 currents only at physiological intracellular Mg(2+) concentrations, and its stimulatory effect was fully abrogated by high internal Mg(2+) levels. Consequently, a TRPM7 variant harboring a gain-of-function mutation was insensitive to further mibefradil activation. Finally, we observed that the effect of mibefradil was selective for TRPM7 when various TRP channels were tested. Taken together, mibefradil acts as a Mg(2+)-regulated agonist of the TRPM7 channel and, hence, uncovers a new class of TRPM7 agonists.

In the lung, acute alveolar hypoxia causes hypoxic pulmonary vasoconstriction (HPV) to maintain ventilation perfusion matching and thus optimal oxygenation of blood. In contrast, global chronic hypoxia triggers a pathological thickening of pulmonary arterial walls, called pulmonary vascular remodelling, leading to persistence of pulmonary hypertension (PH). Moreover, ischaemia or hypoxia can lead to a damage of pulmonary endothelial cells with subsequent oedema formation. Alterations in reactive oxygen species (ROS) have been suggested as a crucial mediator of such responses. Among the various sources of cellular ROS production, NADPH oxidases (NOXs) have been found to contribute to these physiological and pathophysiological signalling processes. NOXs are the only known examples that generate ROS as the primary function of the enzyme system. However, the downstream targets of NOX-derived ROS signalling in hypoxia are still not known. Canonical transient receptor potential (TRPC) channels recently have been recognised as directly or indirectly ROS-activated channels and have been shown to be essential for hypoxia-dependent vascular regulatory processes in the lung. Against this background, we here summarise the current knowledge on NOX-mediated TRPC channel signalling during hypoxia in the pulmonary circulation.

Transient receptor potential cation channel, subfamily M, member 7 (TRPM7) is a cation channel covalently linked to a protein kinase domain. TRPM7 is ubiquitously expressed and regulates key cellular processes such as Mg(2+) homeostasis, motility, and proliferation. TRPM7 is involved in anoxic neuronal death, cardiac fibrosis, and tumor growth. The goal of this work was to identify small molecule activators of the TRPM7 channel and investigate their mechanism of action. We used an aequorin bioluminescence-based assay to screen for activators of the TRPM7 channel. Valid candidates were further characterized using patch clamp electrophysiology. We identified 20 drug-like compounds with various structural backbones that can activate the TRPM7 channel. Among them, the δ opioid antagonist naltriben was studied in greater detail. Naltriben's action was selective among the TRP channels tested. Naltriben activates TRPM7 currents without prior depletion of intracellular Mg(2+) even under conditions of low PIP2. Moreover, naltriben interfered with the effect of the TRPM7 inhibitor NS8593. Finally, our experiments with TRPM7 variants carrying mutations in the pore, TRP, and kinase domains indicate that the site of TRPM7 activation by this small-molecule ligand is most likely located in or near the TRP domain. In conclusion, we identified the first organic small-molecule activators of TRPM7 channels, thus providing new experimental tools to study TRPM7 function in native cellular environments.

Myogenic vasoconstriction is an inherent property of vascular smooth muscle cells (VSMCs) of resistance arteries harboring ill-defined mechanosensing and mechanotransducing elements. G protein-coupled receptors (GPCRs) are discussed as mechanosensors in VSMCs. In this study, we sought to identify and characterize the role and impact of GPCRs on myogenic vasoconstriction. Thus, we analyzed mRNA expression levels of GPCRs in resistance versus preceding conduit arteries revealing a significant enrichment of several GPCRs in resistance vessels. Selective pharmacological blockade of the highly expressed GPCRs in isolated murine mesenteric arteries ex vivo decreased myogenic vasoconstriction. In particular, candesartan and losartan most prominently suppressed myogenic tone, suggesting that AT1 receptors play a central role in myogenic vasoconstriction. Analyzing angiotensinogen(-/-) mice, a relevant contribution of locally produced angiotensin II to myogenic tone could be excluded. Investigation of AT1A (-/-) and AT1B (-/-) murine mesenteric arteries revealed that AT1B, but not AT1A, receptors are key components of myogenic regulation. This notion was supported by examining fura-2-loaded isolated AT1A (-/-) and AT1B (-/-) VSMCs. Our results indicate that in VSMCs, AT1B receptors are more mechanosensitive than AT1A receptors even at comparable receptor expression levels. Furthermore, we demonstrate that the mechanosensitivity of GPCRs is agonist-independent and positively correlates with receptor expression levels.

The luminal composition of the auditory tube influences its function. The mechanisms involved in the monitoring are currently not known. For the lower respiratory epithelium, such a sentinel role is carried out by cholinergic brush cells. Here, using two different mouse strains expressing eGFP under the control of the promoter of choline acetyltransferase (ChAT), we show the presence of solitary cholinergic villin-positive brush cells also in the mouse auditory tube epithelium. They express the vesicular acetylcholine (ACh) transporter and proteins of the taste transduction pathway such as α-gustducin, phospholipase C beta 2 (PLC(β2)) and transient receptor potential cation channel subfamily M member 5 (TRPM5). Immunoreactivity for TRPM5 and PLCβ2 was found regularly, whereas α-gustducin was absent in approximately 15% of the brush cells. Messenger RNA for the umami taste receptors (TasR), Tas1R1 and 3, and for the bitter receptors, Tas2R105 and Tas2R108, involved in perception of cycloheximide and denatonium were detected in the auditory tube. Using a transgenic mouse that expresses eGFP under the promotor of the nicotinic ACh receptor α3-subunit, we identified cholinoceptive nerve fibers that establish direct contacts to brush cells in the auditory tube. A subpopulation of these fibers displayed also CGRP immunoreactivity. Collectively, we show for the first time the presence of brush cells in the auditory tube. These cells are equipped with all proteins essential for sensing the composition of the luminal microenvironment and for communication of the changes to the CNS via attached sensory nerve fibers.

Changes in [Ca(2+)](i) are a central step in platelet activation. In nonexcitable cells, receptor-mediated depletion of intracellular Ca(2+) stores triggers Ca(2+) entry through store-operated calcium (SOC) channels. Stromal interaction molecule 1 (STIM1) has been identified as an endoplasmic reticulum (ER)-resident Ca(2+) sensor that regulates store-operated calcium entry (SOCE), but the identity of the SOC channel in platelets has been controversially debated. Some investigators proposed transient receptor potential (TRP) C1 to fulfil this function based on the observation that antibodies against the channel impaired SOCE in platelets. However, others could not detect TRPC1 in the plasma membrane of platelets and raised doubts about the specificity of the inhibiting anti-TRPC1 antibodies. To address the role of TRPC1 in SOCE in platelets, we analyzed mice lacking TRPC1. Platelets from these mice display fully intact SOCE and also otherwise unaltered calcium homeostasis compared to wild-type. Furthermore, platelet function in vitro and in vivo is not altered in the absence of TRPC1. Finally, studies on human platelets revealed that the presumably inhibitory anti-TRPC1 antibodies have no specific effect on SOCE and fail to bind to the protein. Together, these results provide evidence that SOCE in platelets is mediated by channels other than TRPC1.

Among the classical transient receptor potential (TRPC) subfamily, TRPC1 is described as a mechanosensitive and store-operated channel proposed to be activated by hypoosmotic cell swelling and positive pipette pressure as well as regulated by the filling status of intracellular Ca(2+) stores. However, evidence for a physiological role of TRPC1 may most compellingly be obtained by the analysis of a TRPC1-deficient mouse model. Therefore, we have developed and analyzed TRPC1(-/-) mice. Pressure-induced constriction of cerebral arteries was not impaired in TRPC1(-/-) mice. Smooth muscle cells from cerebral arteries activated by hypoosmotic swelling and positive pipette pressure showed no significant differences in cation currents compared to wild-type cells. Moreover, smooth muscle cells of TRPC1(-/-) mice isolated from thoracic aortas and cerebral arteries showed no change in store-operated cation influx induced by thapsigargin, inositol-1,4,5 trisphosphate, and cyclopiazonic acid compared to cells from wild-type mice. In contrast to these results, small interference RNAs decreasing the expression of stromal interaction molecule 1 (STIM1) inhibited thapsigargin-induced store-operated cation influx, demonstrating that STIM1 and TRPC1 are mutually independent. These findings also imply that, as opposed to current concepts, TRPC1 is not an obligatory component of store-operated and stretch-activated ion channel complexes in vascular smooth muscle cells.

Although chemotaxis has been proposed to guide sperm to egg throughout the animal kingdom, sperm attractants released from mammalian eggs have not been identified. Since the G protein subunit alpha-gustducin is accepted as a marker of chemosensitive cells, attempts were made to explore whether alpha-gustducin is also expressed in spermatozoa of mammals. Immunohistochemical approaches using an anti-alpha-gustducin-specific antibody revealed the most intense immunoreactivity in differentiating spermatids. Further evidence for the alpha-gustducin expression was obtained analyzing testicular and sperm-derived tissue preparations in western blot analyses. To elucidate whether alpha-gustducin is retained in mature spermatozoa, epididymal mouse and rat sperm were subjected to immunocytochemistry as well as immunogold electron microscopy. A specific staining was obtained within the circumference of the midpiece-localized mitochondria, on the axoneme and the outer dense fibers surrounding the microtubules of this region, whereas no labeling was detectable in the end piece regions. The analysis of ejaculated bovine and human sperm revealed a comparable segmental distribution pattern for alpha-gustducin. Although a possible function for alpha-gustducin has yet to be determined, the axonemal-associated localization within the midpiece and principal piece of different mammalian spermatozoa raises the possibility that this G protein alpha-subunit may process intracellular signals controlling sperm motility.

Magnesium is an important cofactor for many biological processes such as protein synthesis, nucleic acid stability and neuromuscular excitability. The extracellular magnesium concentration is regulated tightly by the extent of intestinal absorption and renal excretion. Despite their critical role in magnesium handling, the molecular mechanisms mediating transepithelial transport are still not understood completely. Recently, genetic studies in patients with primary hypomagnesaemia and secondary hypocalcaemia (HSH), a combined defect of intestinal magnesium absorption and renal magnesium conservation, have identified "transient receptor potential (melastatin) 6" (TRPM6) as the first component involved directly in epithelial magnesium reabsorption. TRPM7, the closest homologue of TRPM6, has a central role in Mg(2+) uptake in vertebrate cells since TRPM7-deficient cells become Mg(2+) deficient and are not viable. TRPM7 has been characterized functionally as a constitutively active ion channel permeable for a variety of cations including calcium and magnesium and regulated by intracellular concentrations of magnesium and/or magnesium-nucleotide complexes. Both proteins share the unique feature of cation channels fused to serine/threonine kinase domains. This review summarizes recent data that has emerged from molecular genetic, biochemical and electrophysiological studies on these fascinating two new proteins and their involvement in epithelial magnesium transport.

Among the "classical" or "canonical" transient receptor potential (TRPC) family, the TRPC3, -6, and -7 channels share 75% amino acid identity and are gated by exposure to diacylglycerol. TRPC3, TRPC6, and TRPC7 interact physically and coassemble to form functional tetrameric channels. This review focuses on the TRPC3/6/7 subfamily and describes their functional properties and regulation as homomers obtained from overexpression studies in cell lines. It also summarizes their heteromultimerization potential in vitro and in vivo and presents initial data concerning their physiological functions analyzed in isolated tissues with downregulated channel activity and gene-deficient mouse models.